Antibodies Directed Against the MHC-I Molecule H-2D^d Complexed with an Antigenic Peptide: Similarities to a T Cell Receptor with the Same Specificity

Cell lines

The following cell lines were used: LKD8 (an H-2D^d transfectant of a TAP-defective mouse embryonic cell; Refs. 29, 30); P815 (a DBA/2 mouse mastocytoma); DAP3 (a thymidine kinase-negative L cell, H-2^k, Ref. 31); SKT4.5 (DAP3 cells transfected with H-2D^d; Ref. 32); B4.2.3 (a T cell hybridoma with specificity for peptide P18-I10 bound to H-2D^d; Ref. 32); T2 (a human cell line deficient in TAP1 and TAP2 transporter proteins that expresses low levels of HLA-A2.1 at the cell surface; Refs. 33, 34); EL4 (a murine lymphoma of C57BL origin; Ref. 35); EL4-D^d (EL4 cells transfected with H-2D^d); Jurkat (a human T cell leukemia line; Ref. 36); Jurkat-D^d (Jurkat cells transfected with H-2D^d); and Sp2/0 (a mouse plasmacytoma fusion partner cell line; Ref. 37). EL4-D^d and Jurkat-D^d were the gifts of Drs. S. Hansel and A. Rosenberg, Food and Drug Administration (Bethesda, MD). All cells were maintained in DMEM supplemented with 10% FCS, nonessential amino acids, and glutamine.

Antibodies

The following mAbs were used: 34-5-8S, which recognizes a peptide-dependent conformational epitope on H-2D^d α1α2 domains (38, 39) and W6/32 (40), which binds a conformational epitope on HLA class I molecules.

H-2D^d-restricted viral and self peptides

The following peptides were synthesized by 9-fluoroenylmethoxylcarbonyl chemistry and purified to >95% purity by HPLC at the Laboratory of Molecular and Structural Biology, National Institute of Allergy and Infectious Diseases: P18-I10, RGPGRAFVTI (the HIV IIIB gp160 envelope glycoprotein-derived H-2D^d-restricted peptide; Refs. 32, 41); P18-I10 peptides substituted at different positions (as noted in the figures); H-2D^d motif peptide (42): AGPARAAAL; pD38B: AGPDRTEKAL; pD46A: SGPVALVNFI; pD47: IGPNRAFNF; pNA: CPIRGWAI (residues 77–94 of the influenza virus neuraminidase); and pNP: QPQNGQFIHFY (residues 397–407 of the lymphocytic choriomeningitis virus nucleoprotein).

Proteins

scTCR, an engineered, bacterially expressed, single chain TCR, Vα2.6Vβ7Cβ, with specificity for P18-I10 bound to H-2D^d, has been described in detail previously (19), and the crystal structure of its Vα domain at 2.5 Å resolution has been reported (43). This protein was expressed as inclusion bodies in Escherichia coli strain BL21(DE3) containing the encoding plasmid, solubilized, refolded in vitro, and purified by ion exchange and size exclusion chromatography. Soluble H-2D^d (sH-2D^d) was produced by a transfected L cell line and immunoaffinity purified as described previously (32, 44). sH-2D^d was emptied of self-peptides and loaded with synthetic P18-I10 by a high pH treatment that has been described before (45).

Generation of mAbs specific to H-2D^d/P18-I10 complex

C57BL/6 mice transgenic for a soluble, secreted analog of H-2D^d on a C57BL/6 background (B6.tD^d(α3)/Q10^b, referred to here as DD; Refs. 46, 47) were bred and maintained at the National Institute of Allergy and Infectious Diseases Transgenic Mouse Facility (Frederick, MD). Animals were immunized in one foot pad at 2-wk intervals with H-2D^d/P18-I10 peptide complex (20 μg emulsified in Freund’s complete adjuvant). Following two immunizations, draining lymph node cells were isolated and were fused with SP2/0 cells at a ratio of 2:1 using PEG 4000 (Life Technologies, Grand Island, NY; Ref. 48). After fusion, the cells were resuspended in HAT (hypoxanthine, aminopterin, and thymidine) medium containing 20% FCS and recombinant mouse IL-6 (100 U/ml; Genzyme, Cambridge, MA) at a final concentration of 5 × 10⁵ myeloma cells/ml and were plated at 100 μl/well in 96-well plates. After 10–14 days, supernatant from each well was screened separately by flow cytometry for reactivity with LKD8 cells (H-2D^d-transfected embryonic cells) pulsed with 10 μg/ml P18-I10 peptide in the presence of 5 μg/ml of human β₂-microglobulin (hβ₂m; Fitzgerald Industries International, Concord, MA). Of ∼400 wells examined, five contained Abs that stained LKD8 cells pulsed with P18-I10 peptide. Two of these five Abs did not stain the unloaded LKD8 cells or those pulsed with the control motif peptide (AGPARAAAL). (The protocol in which lymph node rather than spleen cells were used for the fusion was developed after two fusions to spleen in which a total of 1500 hybridomas were screened without identifying any positive clones.) Hybridomas producing Abs specific for the P18-I10/H-2D^d complex were recloned by limiting dilution and retested. The isotyping of mAbs was performed by ELISA using the Mouse MonoAb ID kit (Zymed, San Francisco, CA). (KP14/1 is IgG2b, and KP15/11 is IgG1). The Abs were purified from hybridoma supernatants by affinity chromatography using protein A- and protein G-Sepharose 4 Fast Flow (Pharmacia, Piscataway, NJ) respectively.

Flow cytometry

Approximately 2 × 10⁵ cells were incubated with mAbs for 1 h at 4°C. After washing with PBS containing 2% BSA and 1% sodium azide, cells were incubated with FITC-conjugated goat anti-mouse IgG (1:100 dilution; Dako, Carpinteria, CA) for 40 min at 4°C. After washing in PBS, cell staining (with gating on viable cells using propidium iodide) was assessed with a FACScan flow cytometer, and data were analyzed using Cell Quest software (Becton Dickinson, San Jose, CA).

T cell activation assays

mAbs KP14 and KP15 were tested for their ability to inhibit stimulation of B4.2.3 cells (H-2D^d-restricted T cell hybridoma specific for P18-I10 peptide; Ref. 32). In brief, 1 × 10⁴ cells were stimulated with 2 × 10³ SKT4.5 cells in the presence of graded concentrations of peptide P18-I10, with or without the indicated purified mAbs, for 16 h at 37°C. A portion of the culture supernatant was then removed and tested for IL-2 by ELISA with MiniKit mouse IL-2 (Endogen, Cambridge, MA). The remaining cells were provided with 1 μCi of [³H]thymidine (ICN Pharmaceuticals, Costa Mesa, CA), and incorporation of radiolabel into insoluble material was assessed after a 4-h incubation at 37°C.

Surface plasmon resonance (SPR)

Binding of scTCR (Vα2.6Vβ7Cβ) or purified mAbs (34-5-8S, KP14/1, or KP15/11) to the sH-2D^d complexed with different peptides was evaluated by SPR using a Pharmacia BIAcore (Pharmacia Biosensor AB, Uppsala, Sweden). All binding experiments were performed at 25°C. mAbs were coupled to the biosensor surface through free amino groups at pH 4.5–5.5 according to the conventional method as described previously (45, 49). The scTCR was coupled to the biosensor chip via the thiol group of the free cysteine present in the Cβ domain using a heterobifunctional cross-linking reagent (sulfo-succinimidyl 4[N-maleimido-methyl]cyclohexane-1-carboxylate) as described in detail elsewhere (19, 45). SPR was also used to estimate the affinity of mAb KP14/1 and KP15/11. To determine the kinetic association and dissociation rate constants of MHC/peptide complexes for individual mAbs (KP14/1 and KP15/11), sH-2D^d/P18-I10 complexes were first homogeneously loaded and purified by gel filtration and then injected at different concentrations ranging from 8 × 10⁻⁸ to 6.2 × 10⁻⁶ M over surfaces coupled to mAb KP14/1 or KP15/11. From these kinetic data, kinetic rate constants and the equilibrium constants for dissociation (K_d) were calculated as described previously (19). In addition, K_d values were determined from steady-state binding curves and analyzed according to Scatchard (50). Values obtained from kinetic measurements were consistent with those obtained from the steady-state curves.

Analysis of Ab-variable region gene and encoded protein sequences

mRNA from hybridoma cells producing mAbs KP14/1 or KP15/11 was isolated using the Poly(A)Ttract System 1000 kit (Promega, Madison, WI). These mRNAs served as templates for synthesis of cDNA using hexadeoxyribonucleotides as primers and cloned murine reverse transcriptase. This cDNA was used as a template for preparation of Ab-variable (V) region genes in PCRs, for which heavy and light chain primers were purchased from Pharmacia Biotech and Taq polymerase was purchased from Perkin-Elmer (Norwalk, CT). Amplified heavy and light chain PCR products were purified by 1% agarose gel electrophoresis. For preparation of Fv gene fragments, we used reagents and a protocol provided by Pharmacia Biotech (Mouse ScFv Module). Purified cDNA corresponding to the Ab variable region genes (14V_L, 14V_H, 15V_L, and 15V_H) was inserted into the pCR 2.1 vector (Invitrogen, San Diego, CA) according to the procedure described in the Original TA Cloning Kit, and bacterial transformants were grown on Luria-Bertani agar plates containing kanamycin (50 μg/ml) and X-Gal (5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside) as an indicator. White colonies were picked and grown overnight at 37°C in Luria-Bertani broth containing ampicillin, and plasmid DNA containing the V region fragments was purified (Wizard Minipreps DNA Purification System; Promega) and then sequenced with the Universal M13 Reverse and Forward (-20) Primers (Invitrogen) and the Perkin-Elmer DNA sequencing kit. Products of the sequencing reactions were electrophoresed and analyzed with an ABI 373 DNA sequencer. DNA sequences were analyzed with IgBlast (http://www.ncbi.nlm.nih.gov/igblast/) and the DNA plot analyses of the ImMunoGeneTics database (IMGT) (http://imgt.cines.fr:8104/dnaplot/) (51, 52). Nucleotide sequences have been deposited in GenBank under accession numbers AF261879, AF261880, AF261881, and AF261882. Display of the molecular structure of H-2D^d/P18-I10 using Protein Data Bank (53) coordinates 1DDH (54) was accomplished with GRASP 1.3.6 (55).

CTL induction in vivo and blocking with mAbs

BALB/c mice were immunized i.p. with 5 × 10⁶ PFU recombinant vaccinia virus (vPE16; Ref. 56), which expresses the HIV IIIB gp160 envelope glycoprotein. To block CTL induction against H-2D^d/P18-I10 complexes in vivo, BALB/c mice were injected with different concentrations of KP15 (1 day before, again 4 h before immunization with vPE16, and 4 h, and 1, 2, 3, and 4 days after immunization with vPE16). At each time point, BALB/c mice were injected simultaneously i.p. and i.v. with equal concentrations of KP15. Three weeks later, immune spleen cells were cultured at 5 × 10⁶ per ml in 24-well culture plates in complete T cell medium (RPMI 1640 containing 10% FBS, 2 mM l-glutamine, penicillin (100 U/ml), streptomycin (100 μg/ml), and 5 × 10⁻⁵ M 2-ME). These spleen cells from immunized mice were restimulated in vitro with 4 × 10⁶ of 3300-rad irradiated syngeneic spleen cells pulsed with 1 μM P18-I10 for 7 days before assay. (Additional details of the CTL assay are given elsewhere; Ref. 57 .) The cytolytic activity of these CTL lines was measured in a 4-h ⁵¹Cr release assay. P815 (H-2^d) targets were tested in the presence or absence of P18-I10 peptide (1 μM). For testing the peptide specificity of CTL, ⁵¹Cr-labeled P815 targets were pulsed for 2 h with peptide (1 μM) at the beginning of the assay. The percent specific ⁵¹Cr release was calculated as 100 × (experimental release − spontaneous release)/(maximum release − spontaneous release). Maximum release was determined from supernatants of cells that were lysed by the addition of 5% Triton X-100. Spontaneous release was determined from target cells incubated without added effector cells.

Production of mAbs specific for P18-I10/H-2D^d

To improve the likelihood of obtaining peptide/MHC-restricted mAbs, we developed a strategy based on mice transgenic for a soluble analog of H-2D^d, which we had previously shown to be partially tolerant to the H-2D^d molecule (47). These animals do not readily raise allo-Abs against H-2D^d molecules expressed on lymphoid cells. We prepared soluble H-2D^d molecules which were loaded in vitro with P18-I10 (45) and immunized the transgenic mice. Ab-producing cells were immortalized by somatic cell hybridization (see Materials and Methods), and hybridoma supernatants were screened by flow cytometry in three stages: first, to identify those that reacted with the TAP-deficient H-2D^d-expressing embryonic cell line, LKD8, upon exposure to P18-I10 and human β₂-microglobulin; second, to eliminate those that bound LKD8 without addition of peptide; and third, to eliminate those that bound LKD8 when pulsed with an H-2D^d-motif peptide, a peptide known to bind H-2D^d well, with all nonanchor residues substituted with alanine. Efficient loading of the LKD8 indicator cells with peptide was confirmed by reactivity with 34-5-8S, a mAb that reacts with a structure that is peptide dependent, but not peptide specific (39). Two hybridomas that satisfied our criteria for peptide specificity were identified (KP14 and KP15) that were positive in the first and negative in the second and third flow cytometry screenings by indirect immunofluorescence. They were isolated and immediately cloned by limiting dilution.

The mAbs KP14/1 and KP15/11 showed indirect immunofluorescent staining of high intensity only of LKD8 cells bearing H-2D^d/P18-I10 complexes (Fig. 1⇓A). No reactivity was observed with cells not exposed to peptide or when LKD8 cells were loaded with a motif peptide or other H-2D^d-specific peptides derived from either endogenous proteins (pD38B, pD46A, pD47; Ref. 42) or from viral proteins such as influenza virus neuraminidase (pNA) or the nucleoprotein of lymphocytic choriomeningitis virus (pNP). The failure of the Abs to stain cells pulsed with peptide pD47 (IGPNRAFNF), which is identical in five of nine comparable positions to peptide P18-I10 (RGPGRAFVTI), confirmed the peptide specificity of both mAbs. To evaluate the possibility that the mAbs were specific for P18-I10 alone, even when bound to a different MHC-I molecule, we asked whether either KP14 or KP15 would show specific staining of cells that lacked H-2D^d, but that expressed cell surface MHC-I molecules capable of binding P18-I10. Using T2 cells, a human line expressing HLA-A2, a molecule known to bind P18-I10 (58), we asked whether either Ab bound the peptide/HLA-A2 complex under conditions that allowed a peptide-dependent increase in the binding of the conformation-dependent mAb, W6/32 (Fig. 1⇓B). Although the level of peptide-dependent induction of W6/32 staining was twice the background level, there was no specific induction of reactivity with KP14 or KP15. This observation lends additional support to the view that KP14 and KP15 bind the H-2D^d/P18-I10 complex.

• Download figure
• Open in new tab
• Download powerpoint

FIGURE 1.

Monoclonal Abs KP14/1 and KP15/11 specifically recognize H-2D^d molecules complexed with peptide P18-I10 on LKD8 cells. A, LKD8 cells were pulsed with the indicated peptides and were then stained with mAbs KP14/1 or KP15/11 and then with FITC-conjugated rabbit anti-mouse Ig (bold lines). The cells incubated only with the FITC reagent served as controls (fine lines). LKD8 cells were loaded with the indicated viral (P18-I10, influenza virus neuraminidase, pNP), endogenous (pD38B, pD46A, pD47), or motif peptides and stained for flow cytometry as indicated in detail in Materials and Methods. B, T2 cells expressing HLA-A2.1 were incubated with the indicated mAbs following exposure to either motif or P18-I10 peptide as indicated. Baseline mean cell fluorescence of T2 as assessed with W6/32 was 112.01 and, upon addition of peptide, fluorescence of 174.15 (with P18-I10) and of 179.62 (with H-2D^d motif peptide), a result consistent with previously published findings (58 ,79 ).

Analysis of specificity of binding by SPR

Although these experiments, using cell surface H-2 molecules exposed to P18-I10 and various other peptides, clearly demonstrated the peptide specificity of the Abs, it remained formally possible that the peptide-dependent complexes recognized by these Abs involved some other MHC molecule or even other unrelated molecules expressed on the plasma membrane. To confirm the H-2D^d dependence and the peptide specificity of these Abs, they were tested for their ability to bind purified soluble H-2D^d molecules containing either their pool of self-peptides or to bind preparations that were specifically loaded with P18-I10. These binding studies were performed using SPR as the detection method, and the results are summarized in Fig. 2⇓. Although Ab 34-5-8S binds both the H-2D^d pool (i.e., those molecules complexed with a full repertoire of self peptides) and H-2D^d molecules loaded specifically with P18-I10, mAbs KP14/1 and KP15/11 fail to bind the H-2D^d pool and yet efficiently interact with those H-2D^d molecules specifically loaded with P18-I10. Thus, the purified sH-2D^d protein, in the absence of additional components other than P18-I10, binds these Abs. In addition, using SPR we determined the affinities of the two Abs for H-2D^d/P18-I10 complexes. KP14/1 and KP15/11 had affinity constants, K_d, of 7.14 × 10⁻⁷ and 8.47 × 10⁻⁷ M, respectively.

• Download figure
• Open in new tab
• Download powerpoint

FIGURE 2.

Binding of sH-2D^d/P18-I10 complexes to mAb KP14/1 and KP15/11. Direct binding of these complexes to the indicated Abs was assessed by SPR. Purified Abs (KP14/1, KP15/11, and the anti-H-2D^d mAb, 34-5-8S), were coupled through amino groups to a biosensor chip as described in Materials and Methods. Purified sH-2D^d containing its natural repertoire of self-peptides as expressed by an L cell transfectant (D^d) and sH-2D^d loaded with P18-I10 (D^d+P18-I10) were exposed to separate biosensor surfaces coupled to the indicated Abs. The injection of the solution phase sH-2D^d/P18-I10 complexes was initiated at t = 80 s, and the buffer washout phase began at t = 210 s.

Both mAbs KP14/1 and KP15/11 were obtained from the same fusion, in which lymph nodes from four immunized mice were pooled. Even though the clones producing these Abs were detected in two different 96-well plates, we could not exclude the possibility that these clones were siblings. The difference in Ab isotypes (KP14/1 is IgG2b and KP15/11 is IgG1) did not completely exclude this possibility because a common V region might switch independently to several C regions in the early stabilization of the fusion (59). In addition, binding experiments using SPR revealed that KP14/1 and KP15/11 competed for the same binding site on H-2D^d complexes (data not shown). Therefore, we studied the fine specificity of both Abs using a panel of synthetic P18-I10-related peptides and several different H-2D^d-positive cell lines, including the TAP-negative H-2D^d transfectant LKD8. Immunofluorescence with the two Abs revealed that several different peptide/H-2D^d complexes were recognized distinctly. In the examples illustrated here (Fig. 3⇓), the G6-substituted peptide formed a complex recognized better by KP14/1 than by KP15/11. The M1- and L1-peptide/H-2D^d complexes were bound better by KP15/11 than by KP14/1, and in some experiments L1/H-2D^d complexes were not recognized at all by KP14/1. Despite the fine specificity differences between the two mAbs, the relative level of staining of cells pulsed with different peptides was independent of the cell line studied.

• Download figure
• Open in new tab
• Download powerpoint

FIGURE 3.

Differences in fine specificities between mAbs KP14/1 and KP15/11 are reflected in differences in binding intensity to peptide/H-2D^d complexes made with some P18-I10 variants. LKD8, SKT4.5, or P815 cells were pulsed with P18-I10 variants (G6 (RGPGRGFVTI), M1 (MGPGRAFVTI), or L1 (LGPGRAFVTI)) followed by staining with mAb KP14/1 or KP15/11. The complex G6/H-2D^d is better recognized with mAb KP14/1 (dashed line) than with mAb KP15/11 (solid line). The opposite observation was made with variants M1 and L1. Cells stained with FITC-secondary Ab alone served as a negative control (dotted line).

We then surveyed of a panel of 27 different substituted peptides using the H-2D^d-transfected L cell SKT4.5 as the indicator (Fig. 4⇓). As seen in Fig. 4⇓A, with the exception of peptides M1, L1, G6 (as pointed out above), and A2, the majority of the synthetic peptide variants generate peptide/MHC complexes that are seen equivalently by both KP14/1 and KP15/11. The motif peptide (AGPARAAAL) failed to generate a complex capable of being recognized well by either of the Abs, a behavior similar to that of peptides substituted at positions 5, 6, and 7 (with the exception of the strong recognition by KP14/1 of the G6 substitution). Peptides L1, M1, and A2 were seen better by KP15/11. The pattern of binding as revealed by immunofluorescence of live cells was closely mimicked by the pattern of binding as determined in a direct assay using purified proteins and SPR (Fig. 4⇓B).

• Download figure
• Open in new tab
• Download powerpoint

FIGURE 4.

Different peptide/MHC complexes are bound distinctly by mAbs KP14/1 and KP15/11 as compared with a single chain TCR. A, SKT4.5 cells were pulsed with no peptide, P18-I10, or each of the indicated variant synthetic peptides and then analyzed by flow cytometry for mean fluorescence index (mean FI) with mAbs KP14/1 (open columns) or KP15/11 (filled columns). B, Binding was assessed by SPR for binding of the indicated peptide/H-2D^d complexes to either KP14/1 (open columns) or KP15/11 (filled columns); or SPR for binding of the indicated peptide/MHC complexes to immobilized scTCR (C).

We have previously described a direct assay in which the binding of a biotinylated recombinant three-domain TCR to cells exposed to peptides closely paralleled the pattern of T cell activation induced by the same panel of peptides (19). Here we compared the direct binding of Abs KP14/1 and KP15/11 to peptide complexed with H-2D^d with the binding of the scTCR (Fig. 4⇑C). Like mAbs KP14/1 and KP15/11, the scTCR failed to bind to peptide/MHC complexes formed with variant peptides having amino acid substitutions in positions 5, 7, and 10. In addition, the scTCR was more sensitive than the mAbs to substitutions at positions 8 and 9. In addition, the scTCR bound well to the Y7 peptide/H-2D^d complex, whereas neither of the mAbs did. Thus, although the Abs bind the same peptide/MHC complex as the scTCR, their fine specificities differ from each other and from that of the scTCR. The suggestion that the two clones do not express identical Ab heavy and light chain V regions has been confirmed by the analysis of their nucleotide sequences (see below).

Inhibition of T cell recognition by mAbs KP14/1 and KP15/11

To compare the nature of the site to which the Abs and TCR bind, the two Abs were used to inhibit the peptide dose-dependent activation of the B4.2.3 T cell hybridoma (see Fig. 5⇓). As shown in A, in the absence of added Ab, the hybridoma is stimulated to release IL-2 or to be inhibited in its constitutive proliferation at a dose of P18-I10 of ∼1 nM. Ab KP15/11 (0.3 mg/ml) potently inhibits IL-2 secretion and constitutive proliferation, even at the highest doses of peptide tested (5 nM). The IL-2 secretion assay appears to be more sensitive than the proliferation inhibition one to the effect of the mAbs. Abs KP14/1 (0.3 mg/ml) and the anti-α1α2 domain-dependent mAb, 34-5-8S (0.3 mg/ml), inhibited less well. At a lower dose (0.06 mg/ml), KP14/1 showed no significant inhibition of the peptide-dependent activation. The above results are all consistent with the view that these two peptide-specific, MHC-restricted mAbs bind a site that overlaps that seen by the TCR with the same specificity.

• Download figure
• Open in new tab
• Download powerpoint

FIGURE 5.

Inhibition of peptide presentation by MHC/peptide-specific mAbs. SKT4.5 H-2D^d transfectant cells were pulsed with the indicated concentrations of P18-I10 and then exposed to B4.2.3 cells in the presence of the indicated mAbs as described in detail in Materials and Methods. Stimulation was assessed by measurement of IL-2 produced or by [³H]thymidine incorporation.

Peptide/MHC-specific mAbs block the induction of CTL in vivo

The above assays indicated that KP14 and KP15 mAbs both recognize P18-I10/H-2D^d complexes produced from purified protein and synthetic peptide. However, they do not address the issue as to whether the mAbs bind peptide generated via the endogenous MHC-I presentation pathway. To demonstrate the effectiveness of the binding of such mAbs to peptides generated in this way, we administered KP15 mAb to BALB/c animals immediately before and during exposure to a dose of recombinant vaccinia virus encoding the entire HIV IIIB gp160 envelope glycoprotein. A schedule of seven injections of mAb was used, and the dose per injection was evaluated. Three weeks after in vivo priming, spleen cells were removed and restimulated in culture with the antigenic peptide for 1 wk. The resulting CTL were evaluated for lysis of peptide-pulsed target cells (Fig. 6⇓). As shown in Fig. 6⇓, exposure of the host animal during priming with a dose of 0.001 mg/ml of KP15 had no significant effect in reducing the activity of bulk CTL as compared with an isotype-matched IgG1 control. However, doses of 0.01, 0.1, and 1 mg/ml showed significant inhibition of the resultant CTL. This experiment demonstrates that the in vivo use of the mAb can directly block the priming of CTL in vivo as elicited by infection with the recombinant vaccinia vector.

• Download figure
• Open in new tab
• Download powerpoint

FIGURE 6.

Inhibition of CTL priming by mAb KP15. BALB/c mice were immunized with recombinant vaccinia virus expressing HIV IIIB gp-160 envelope glycoprotein (vPE16) and different doses of purified mAb KP15 or an isotype-matched control mAb according to the schedule given in Materials and Methods. A, Data expressed as a function of E:T ratio. Doses used were: ▪, 0.001 mg/ml; ▴, 0.01 mg/ml; □, 0.1 mg/ml; ▵, 1.0 mg/ml. ×, P815 target cells not pulsed with peptide tested with vPE16-primed effectors. ○, Cytolysis of cells raised in the presence of 1.0 mg/ml of an isotype-matched control mAb. B, The same data plotted as a function of KP15 concentration for the different E:T ratios (○, 100:1; □, 50:1; •, 25:1; and ▪, 12:1).

Structural comparison of mAbs and TCR

One of our initial goals in isolating these mAbs was to gain insight into the structural basis by which molecules of the Ig superfamily, mAb, and cloned TCR interact specifically with a peptide/MHC complex. To explore the relationship among the three molecules, KP14, KP15, and the Vα2.6Vβ7Cβ scTCR of hybridoma B4.2.3, we compared their amino acid sequences. We compared the V_H sequences of KP14 and KP15 with the Vβ sequence of the B4.2.3 TCR and the V_L sequences of KP14 and KP15 with Vα sequences of the TCR (Fig. 7⇓). Structural comparisons of Vα and Vβ have not yet definitively resolved the issue of which is more similar to V_H or V_L, but V_H and Vβ are both assembled at the DNA level from V, D, and J gene segments, and Vα generally appears to provide a better fit when aligned with V_L (13). For these TCR-like mAbs, the V_H showed greater similarity to TCR Vβ, and V_L to Vα. Sequence comparisons reveal intriguing similarities among these molecules (see Fig. 7⇓ and Table I⇓). First, both V_H, although they differed in CDR3, used the identical germline V gene. This is the mouse IGHV3 K01569 according to the IMGT nomenclature and is designated V_H36-60* by IgBLAST (Table I⇓). The CDR3 of the heavy chain of KP14 (labeled 14H) is 12 residues long and that of KP15 (15H) is 11, whereas that of the Vβ of the B4.2.3 TCR is 10 (Fig. 7⇓). The two mAb heavy chains use different IGHD segments (M35332 IGHD-FL16.1*02 for 14H and L32868 IGHD-Q52*01 for 15H). The J_H segments used for these two heavy chain genes seem to be the same (V00880 IGHJ2*01). These heavy chain CDR3s are similar in that they each contain three acidic residues and four aromatic ones. The amino acid sequence alignment of the heavy chains with the Vβ (Fig. 7⇓) is also remarkable for the identity of residues 50–54 of the heavy chains with residues 47–51 of the Vβ (amino acid residues YISYD). This sequence coincides with CDR2 and may be indicative of an important role CDR2 plays in a specific interaction with H-2D^d.

• Download figure
• Open in new tab
• Download powerpoint

FIGURE 7.

Comparison of amino acid sequences of mAb KP14 and KP15 heavy and light chains. The V regions of the two mAb heavy and light chains were cloned and sequenced as described in Materials and Methods. In A, the heavy chains are aligned with the Vβ of clone B4.2.3 that was previously reported. In B, the light chains are compared with the Vα sequence.

Analysis of the two mAb light chains (Fig. 7⇑B and Table I⇑) reveals that they are distinct, though they both belong to the mouse κ light chain V family (60). They represent different germline genes: IGKV10S1 and IGKV6S6 according to the IMGT nomenclature (51, 52), and use different Jκ segments. These two different κ light chains apparently represent structurally alternative solutions for the stringent dual requirements of productively interacting with their respective heavy chains and of binding the P18-I10/H-2D^d complex.